Compounds and compositions for delivering active agents

ABSTRACT

Carrier compounds, compositions, and dosage unit forms therefor which are useful in the delivery of active agents are provided. The present invention provides a compound having the formula: ##STR1## or a salt thereof, wherein the compound may be used in a composition or dosage unit form for delivery of at least one active agent, including a peptide, mucopolysaccharide, carbohydrate, or a lipid. Methods of administration and preparation of the compounds and compositions of the invention are provided as well, including oral administration. Further, the compositions of the invention may be prepared by mixing at least one active agent, at least one carrier compound, and, optionally, a dosing vehicle.

This application is a continuation of PCT/US96/04580, filed Apr. 1,1996, which is a Continuation-In-Part of application Ser. No.08/414,654, now U.S. Pat. No. 5,650,386, filed Mar. 31, 1995 and whichclaims benefit to application Ser. No. 60/003,111, filed Sep. 1, 1995,and application Ser. No. 60/017,902, filed Mar. 29, 1996.

FIELD OF THE INVENTION

The present invention relates to compounds for delivering active agents,and particularly biologically or chemically active agents such as, forexample, bioactive peptides and the like. These compounds are used ascarriers to facilitate the delivery of a cargo to a target. The carriersare modified amino acids and are well suited to form non-covalentmixtures with biologically-active agents for oral administration toanimals. Methods for the preparation and for the administration of suchcompositions are also disclosed.

BACKGROUND OF THE INVENTION

Conventional means for delivering active agents are often severelylimited by biological, chemical, and physical barriers. Typically, thesebarriers are imposed by the environment through which delivery occurs,the environment of the target for delivery, or the target itself.

Biologically or chemically active agents are particularly vulnerable tosuch barriers. For example in the delivery to animals of pharmacologicaland therapeutic agents, barriers are imposed by the body. Examples ofphysical barriers are the skin and various organ membranes that must betraversed before reaching a target. Chemical barriers include, but arenot limited to, pH variations, lipid bi-layers, and degrading enzymes.

These barriers are of particular significance in the design of oraldelivery systems. Oral delivery of many biologically or chemicallyactive agents would be the route of choice for administration to animalsif not for biological, chemical, and physical barriers such as varyingpH in the gastro-intestinal (GI) tract, powerful digestive enzymes, andactive agent impermeable gastro-intestinal membranes. Among the numerousagents which are not typically amenable to oral administration arebiologically or chemically active peptides, such as calcitonin andinsulin; polysaccharides, and in particular mucopolysaccharidesincluding, but not limited to, heparin; heparinoids; antibiotics; andother organic substances. These agents are rapidly rendered ineffectiveor are destroyed in the gastrointestinal tract by acid hydrolysis,enzymes, or the like.

Earlier methods for orally administering vulnerable pharmacologicalagents have relied on the co-administration of adjuvants (e.g.,resorcinols and non-ionic surfactants such as polyoxyethylene oleylether and n-hexadecylpolyethylene ether) to increase artificially thepermeability of the intestinal walls, as well as the co-administrationof enzymatic inhibitors (e.g., pancreatic trypsin inhibitors,diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymaticdegradation.

Liposomes have also been described as drug delivery systems for insulinand heparin. See, for example, U.S. Pat. No. 4,239,754; Patel et al.(1976), FEBS Letters, Vol. 62, pg. 60; and Hashimoto et al. (1979),Endocrinology Japan, Vol. 26, pg. 337.

However, broad spectrum use of such drug delivery systems is precludedbecause: (1) the systems require toxic amounts of adjuvants orinhibitors; (2) suitable low molecular weight cargos, i.e. activeagents, are not available; (3) the systems exhibit poor stability andinadequate shelf life; (4) the systems are difficult to manufacture; (5)the systems fail to protect the active agent (cargo); (6) the systemsadversely alter the active agent; or (7) the systems fail to allow orpromote absorption of the active agent.

More recently, microspheres of artificial polymers of mixed amino acids(proteinoids) have been used to deliver pharmaceuticals. For example,U.S. Pat. No. 4,925,673 describes drug-containing proteinoid microspherecarriers as well as methods for their preparation arid use. Theseproteinoid microspheres are useful for the delivery of a number ofactive agents.

There is still a need in the art for simple, inexpensive deliverysystems which are easily prepared and which can deliver a broad range ofactive agents.

SUMMARY OF THE INVENTION

Compositions which are useful in the delivery of active agents areprovided. These compositions include at least one active agent, andpreferably a biologically or chemically active agent, and at least oneof the following compounds I-CXXIII, or salts thereof.

    __________________________________________________________________________     ##STR2##                                         I     ##STR3##                                         II     ##STR4##                                         III     ##STR5##                                         IV     ##STR6##                                         V     ##STR7##                                         VI     ##STR8##                                         VII     ##STR9##                                         VIII     ##STR10##                                        IX     ##STR11##                                        X     ##STR12##                                        XI     ##STR13##                                        XII     ##STR14##                                        XIII     ##STR15##                                        XIV     ##STR16##                                        XV     ##STR17##                                        XVI     ##STR18##                                        XVII     ##STR19##                                        XVIII     ##STR20##                                        XIX     ##STR21##                                        XX     ##STR22##                                        XXI     ##STR23##                                        XXII     ##STR24##                                        XXIII     ##STR25##                                        XXIV     ##STR26##                                        XXV     ##STR27##                                        XXVI     ##STR28##                                        XXVII     ##STR29##                                        XXVIII     ##STR30##                                        XXIX     ##STR31##                                        XXX     ##STR32##                                        XXXI     ##STR33##                                        XXXII     ##STR34##                                        XXXIII     ##STR35##                                        XXXIV     ##STR36##                                        XXXV     ##STR37##                                        XXXVI     ##STR38##                                        A    Compound       n m                   X    __________________________________________________________________________    XXXVII         0 0                   4-Cl    XXXVIII        3 0                   H    XXXIX          3 1                   4-CH.sub.3    XL             3 1                   2-F    XLI            3 1                   2-CH.sub.3    XLII           3 0                   3-CF.sub.3    XLIII          3 4                   H    XLIV           3 0                   3-Cl    XLV            3 0                   3-F    XLVI           3 0                   3-CH.sub.3    XLVII          0 0                   2-CF.sub.3    XLVIII         1 2                   H    XLIX           3 2                   2-F    L              3 0                   3,4-OCH.sub.2 O    LI             3 0                   2-COOH    LII            1 0                   2-OH    LIII           3 0                   2,6-dihydroxy    LIV            2 0                   2-OH    LV             0 0                   2,4-difluoro    LVI            2 0                   2,6-dihydroxy    LVII           0 0                   4-CF.sub.3    LVIII          3 0                   3-NMe.sub.2    LIX            2 0                   3-NMe.sub.2    LX             3 0                   2,6-dimethyl    LXI            3 0                   2-NO.sub.2    LXII           3 0                   2-CF.sub.3    LXIII          3 0                   4-n-Pr    LXIV           3 0                   2-NH.sub.2    LXV            3 0                   2-OCH.sub.3    LXVI           3 0                   3-NO.sub.2    LXVII          3 0                   3-NH.sub.2    LXVIII         2 0                   2-NO.sub.2    LXIX           2 0                   2-NH.sub.2    LXX            3 0                   2-OCF.sub.3    LXXI           2 0                   2-OCH.sub.3    LXXII          2 0                   2-OCF.sub.3    __________________________________________________________________________     ##STR39##                                        B    Compound          n                           X    __________________________________________________________________________    LXXIII          3                           4-CF.sub.3    LXXIV 1                           2-F    LXXV  1                           4-CF.sub.3    LXXVI 3                           3,4-dimethoxy    LXXVII          0                           3-OCH.sub.3    LXXVIII          3                           3-OCH.sub.3    LXXIX 3                           2,6-difluoro    LXXX  3                           4-CH.sub.3    LXXXI 1                           4-OCH.sub.3    LXXXII          2                           2-F    LXXXIII          0                           2-F    LXXXIV          2                           4-OCH.sub.3    LXXXV 0                           2-OCH.sub.3    LXXXVI          2                           2-OCH.sub.3    LXXXVII          0                           4-CF.sub.3    LXXXVIII          3                           3-F    LXXXIX          3                           2-OCH.sub.3    __________________________________________________________________________     ##STR40##                                        C    Compound       n m                  X    __________________________________________________________________________    XC             3 0                  2-carboxycyclohexyl    XCI            3 3                  cyclohexyl    XCII           3 0                  2-adamantyl    XCIII          3 0                  1-morpholino    __________________________________________________________________________     ##STR41##                                        D    Compound          m    __________________________________________________________________________    XCIV  0    XCV   3    __________________________________________________________________________     ##STR42##                                        E    Compound          X    __________________________________________________________________________    XCVI  OH    XCVII O    __________________________________________________________________________     ##STR43##                                        F    Compound          n    __________________________________________________________________________    XCVIII          0    XCIX  2    __________________________________________________________________________     ##STR44##                                        C     ##STR45##                                        CI     ##STR46##                                        CII     ##STR47##                                        CIII     ##STR48##                                        CIV     ##STR49##                                        CV     ##STR50##                                        CVI     ##STR51##                                        CVII     ##STR52##                                        CVIII     ##STR53##                                        CIX     ##STR54##                                        CX     ##STR55##                                        G    Compound       n m                     X    __________________________________________________________________________    CXI            6 0                     2-OH    CXII           7 3                     H    CXIII          7 0                     2-I    CXIV           7 0                     2-Br    CXV            7 0                     3-NO.sub.2    CXVI           7 0                     3-N(CH.sub.3).sub.2    CXVII          7 0                     2-NO.sub.2    CXVIII         7 0                     4-NO.sub.2    CXIX           9 0                     2-OH    __________________________________________________________________________     ##STR56##                                        H    Compound                 X    __________________________________________________________________________    CXX                      1-morpholino    CXXI                     O-t-Butyl    CXXII                    CH(CH.sub.2 Ph)NC(O)O-t-Bu    CXXIII                   2-hydroxyphenyl    __________________________________________________________________________

It has been discovered that organic acid compounds, and their salts,having an aromatic amide group, having a hydroxy group substituted inthe ortho position on the aromatic ring, and a lipophilic chain withfrom about 4 carbon atoms to about 20 atoms in the chain are useful ascarriers for the delivery of active agents. In a preferred form thelipophilic chain can have from 5 to 20 carbon atoms.

Compositions comprising the carrier compounds discussed above and activeagents have been shown effective in delivering active agents to selectedbiological systems. These compositions include at least one active agentwhich is preferably a biologically or chemically active agent, and atleast one carrier compound having the formula

    2--HO--Ar--CONR.sup.8 --R.sup.7 --COOH

wherein

Ar is a substituted or unsubstituted phenyl or naphthyl;

R⁷ is selected from the group consisting of C₄ to C₂₀ alkyl, C₄ to C₂₀alkenyl, phenyl, naphthyl, (C₁ to C₁₀ alkyl) phenyl, (C₁ to C₁₀ alkenyl)phenyl, (C₁ to C₁₀ alkyl) naphthyl, (C₁ to C₁₀ alkenyl) naphthyl, phenyl(C₁ to C₁₀ alkyl), phenyl (C₁ to C₁₀ alkenyl), naphthyl (C₁ to C₁₀alkyl), and naphthyl (C₁ to C₁₀ alkenyl);

R⁸ is selected from the group consisting of hydrogen, C₁ to C₄ alkyl, C₁to C₄ alkenyl, hydroxy, and C₁ to C₄ alkoxy:

R⁷ is optionally substituted with C₁ to C₄ alkyl, C₁ to C₄ alkenyl, C₁to C₄ alkoxy, --OH, --SH and --CO₂ R⁹ or any combination thereof;

R⁹ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl;

R⁷ is optionally interrupted by oxygen, nitrogen, sulfur or anycombination thereof;

with the proviso that the compounds are not substituted with an aminogroup in the position alpha to the acid group, or salts thereof.

The preferred R⁶ groups are of C₄ to C₂₀ alkyl and C₄ to C₂₀ alkenyl.The most preferred R⁶ groups are C₅ to C₂₀ alkyl and C₅ to C₂₀ alkenyl.

A preferred carrier compound can have the formula ##STR57## wherein R⁷is defined above.

Further contemplated by the present invention are dosage unit forms thatinclude these compositions.

Also contemplated is a method for preparing these compositions whichcomprises mixing at least one active agent with at least one compound asdescribed above, and optionally, a dosing vehicle.

In an alternative embodiment, these non-toxic compounds are orallyadministered to animals as part of a delivery system by blending ormixing the compounds with an active agent prior to administration.

Further provided is a method for the preparation of a compound havingthe formula ##STR58## wherein Y is ##STR59## or SO₂ ; R¹ is C₃ -C₂₄alkyl, C₂ -C₂₀ alkenyl, C₂ -C₂₀ alkyne, cycloalkyl, or aromatic;

R² is hydrogen, C₁ -C₄ alkyl, or C₂ -C₄ alkenyl; and

R³ is C₁ -C₇ alkyl, C₃ -C₁₀ cycloalkyl, aryl, thienyl, pyrrolo, orpyridyl, where R³ is optionally substituted by-one or more C₁ -C₅ alkylgroup, C₂ -C₄ alkenyl group, F, Cl, OH, SO₂, COOH, or SO₃ H;

said method comprising

(a) reacting in water and the presence of a base, a compound having theformula ##STR60## with a compound having the formula

    R.sup.3 --Y--X,

wherein

Y, R¹, R², and R³ are as above and X is a leaving group.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic illustration of the results of subcutaneousinjection of rhGH composition in rats.

FIG. 2 is a graphic illustration of the results of Sublingual (SL),intranasal (IN), and intracolonic (IC) dosing of rhGH in rats.

FIG. 3 is a graphic illustration of the results of intracolonic dosingof delivery of heparin with compound XXXI carrier.

DETAILED DESCRIPTION OF THE INVENTION

The specific compositions of the present invention include an activeagent and a modified amino acid. These compositions may be used todeliver various active agents through various biological, chemical, andphysical barriers and are particularly suited for delivering activeagents which are subject to environmental degradation. The compositionsof the subject invention are particularly useful for delivering oradministering biologically or chemically active agents to any animalssuch as birds; mammals, such as - primates and particularly humans; andinsects.

Other advantages of the present invention include the use of easy toprepare, inexpensive raw materials. The compositions and the formulationmethods of the present invention are cost effective, simple to perform,and amenable to industrial scale up for commercial production.

Subcutaneous, sublingual, and intranasal coadministration of an activeagent, such as recombinant human growth hormone (rhGH), and the deliveryagents, and particularly proteins, described herein results in anincreased bioavailability of the active agent compared to administrationof the active agent alone. A similar result is obtained bycoadministration of salmon calcitonin with the delivery agents, in rats.Data supporting these findings are presented in the examples.

Active Agents

Active agents suitable for use in the present invention includebiologically or chemically active agents, chemically active agents,including, but not limited to, fragrances, as well as other activeagents such as, for example, cosmetics.

Biologically or chemically active agents include, but are not limitedto, pesticides, pharmacological agents, and therapeutic agents. Forexample, biologically or chemically active agents suitable for use inthe present invention include, but are not limited to, peptides, andparticularly small peptides; hormones, and particularly hormones whichby themselves do not or only a fraction of the administered dose passesthrough the gastro- intestinal mucosa and/or are susceptible to chemicalcleavage by acids and enzymes in the gastrointestinal tract;polysaccharides, and particularly mixtures of muco-polysaccharides;carbohydrates; lipids; or any combination thereof. Further examplesinclude, but are not limited to, human growth hormones; bovine growthhormones; growth releasing hormones; interferons; interleukin-1;insulin; heparin, and particularly low molecular weight heparin;calcitonin; erythropoietin; atrial naturetic factor; antigens;monoclonal antibodies; somatostatin; adrenocorticotropin, gonadotropinreleasing hormone; oxytocin; vasopressin; cromolyn sodium (sodium ordisodium chromoglycate); vancomycin; desferrioxamine (DFO); parathyroidhormone anti-microbials, including, but not limited to anti-fungalagents; or any combination thereof.

Modified Amino Acids

The terms modified amino acid, modified poly amino acid, and modifiedpeptide are meant to include amino acids which have been modified, orpoly amino acids and peptides in which at least one amino acid has beenmodified, by acylating or sulfonating at least one free amine group withan acylating or sulfonating agent which reacts with at least one of thefree amine groups present.

Amino acids, poly amino acids, and peptides, in modified form, may beused to deliver active agents including, but not limited to,biologically or chemically active agents such as for example,pharmacological and therapeutic agents.

An amino acid is any carboxylic acid having at least one free aminegroup and includes naturally occurring and synthetic amino acids.

Poly amino acids are either peptides or two or more amino acids linkedby a bond formed by other groups which can be linked, e.g. an ester,anhydride, or an anhydride linkage.

Peptides are two or more amino acids joined by a peptide bond.

Peptides can vary in length from dipeptides with two amino acids to polypeptides with several hundred amino acids. See Chambers BiologicalDictionary, editor Peter M. B. Walker, Cambridge, England: ChambersCambridge, 1989, page 215. Special mention is made of di-peptides,tri-peptides, tetra-peptides, and penta-peptides.

Although compounds I-CXXIII above have been found to act as carriers forthe oral delivery of biologically or chemically active agents, specialmention is made of compounds I-XXXI above.

Modified amino acids are typically prepared by modifying the amino acidor an ester thereof. Many of these compounds are prepared by acylationor sulfonation with agents having the formula

    X--Y--R.sup.4

wherein:

R⁴ is the appropriate radical to yield the modification indicated in thefinal product,

Y is ##STR61## or SO₂, and X is a leaving group. Typical leaving groupsinclude, but are not limited to, halogens such as, for example,chlorine, bromine, and iodine. Additionally, the correspondinganhydrides are modifying agents.

Many of the compounds of the present invention can be readily preparedfrom amino acids by methods within the skill of those in the art basedupon the present disclosure. For example, compounds I-VII are derivedfrom aminobutyric acid; Compounds VIII-X and XXXII-XXXV are derived fromaminocaproic acid; and Compounds XI-XXVI and XXXVI are derived fromaminocaprylic acid. For example, the modified amino acid compounds abovemay be prepared by reacting the single amino acid with the appropriatemodifying agent which reacts with free amino moiety present in the aminoacids to form amides. Protecting groups may be used to avoid unwantedside reactions as would be known to those skilled in the art.

The amino acid can be dissolved in aqueous alkaline solution of a metalhydroxide, e.g., sodium or potassium hydroxide, and heated at atemperature ranging between about 5° C. and about 70° C., preferablybetween about 10° C. and about 40° C., for a period ranging betweenabout 1 hour and about 4 hours, preferably about 2.5 hours. The amountof alkali employed per equivalent of NH₂ groups in the amino acidgenerally ranges between about 1.25 and about 3 mmole, preferablybetween about 1.5 and about 2.25 mmole per equivalent of NH₂. The pH ofthe-solution generally ranges between about 8 and about 13, preferablyranging between about 10 and about 12.

Thereafter, the appropriate amino modifying agent is added to the aminoacid solution while stirring. The temperature of the mixture ismaintained at a temperature generally ranging between about 5° C. andabout 70° C., preferably between about 10° C. and about 40° C., for aperiod ranging between about 1 and about 4 hours. The amount of aminomodifying agent-employed in relation to the quantity of amino acid isbased on the moles of total free NH₂ in the amino acid. In general, theamino modifying agent is employed in an amount ranging between about 0.5and about 2.5 mole equivalents, preferably between about 0.75 and about1.25 equivalents, per molar equivalent of total NH₂ group in the aminoacid.

The reaction is quenched by adjusting the pH of the mixture with asuitable acid, e.g., concentrated hydrochloric acid, until the pHreaches between about 2 and about 3. The mixture separates on standingat room temperature to form a transparent upper layer and a white oroff-white precipitate. The upper layer is discarded, and the modifiedamino acid is collected from the lower layer by filtration ordecantation. The crude modified amino acid is then dissolved in water ata pH ranging between about 9 and about 13, preferably between about 11and about 13. Insoluble materials are removed by filtration and thefiltrate is dried in vacuo. The yield of modified amino acid generallyranges between about 30 and about 60%, and usually about 45%.

If desired, amino acid esters, such as, for example benzyl, methyl, orethyl esters of amino acid compounds, may be used to prepare themodified amino acids of the invention. The amino acid ester, dissolvedin a suitable organic solvent such as dimethylformamide, pyridine, ortetrahydrofuran is reacted with the appropriate amino modifying agent ata temperature ranging between about 5° C. and about 70° C., preferablyabout 25° C., for a period ranging between about 7 and about 24 hours.The amount of amino modifying agent used relative to the amino acidester is the same as described above for amino acids. This reaction maybe carried out with or without a base such as, for example,triethylamine or diisopropylethylamine.

Thereafter, the reaction solvent is removed under negative pressure andthe ester functionality is removed by hydrolyzing the modified aminoacid ester with a suitable alkaline solution, e.g. 1N sodium hydroxide,at a temperature ranging between about 50° C. and about 80° C.,preferably about 70° C., for a period of time sufficient to hydrolyzeoff the ester group and form the modified amino acid having a freecarbox-yl group. The hydrolysis mixture is then cooled to roomtemperature and acidified, e.g. aqueous 25% hydrochloric acid solution,to a pH ranging between about 2 and about 2.5. The modified amino acidprecipitates out of solution and is recovered by conventional means suchas filtration or decantation. Benzyl esters may be removed byhydrogenation in an organic solvent using a transition metal catalyst.

The modified amino acid may be purified by recrystallization or byfractionation on solid column supports. Suitable recrystallizationsolvent systems include acetonitrile, methanol and tetrahydrofuran.Fractionation may be performed on a suitable solid column supports suchas alumina, using methanol/n-propanol mixtures as the mobile phase;reverse phase column supports using trifluoroacetic acid/acetonitrilemixtures as the mobile phase; and ion exchange chromatography usingwater as the mobile phase. When anion exchange chromatography isperformed, preferably a subsequent 0-500 mM sodium chloride gradient isemployed.

In an alternate method modified amino acids having the formula II##STR62## wherein Y is ##STR63## or S0₂ ; R¹ is C₃ -C₂₄ alkyl, C₂ -C₂₀alkenyl, C₂ -C₂₀ alkyne, cycloalkyl, or aromatic;

R² is hydrogen, C₁ -C₄ alkyl, or C₂ -C₄ alkenyl; and

R³ is C₁ -C₇ alkyl, C₃ -C₁₀ cycloalkyl, aryl, thienyl, pyrrolo, orpyridyl, where R³ is optionally substituted by one or more C₁ -C₅ alkylgroup, C₂ -C₄ alkenyl group, F, Cl, OH, SO₂, COOH or, SO₃ H; may beprepared by

(a) reacting in water and the presence of a base a compound having theformula ##STR64## with a compound having the formula

    R.sup.3 --Y--X,

wherein

Y, R¹, R², and R³ are as above and X is a leaving group.

Compound CXXV can be prepared, for example by the method described inOlah et al., Synthesis, 537-538 (1979).

Compound XXXI was prepared as described in Scheme I from10-undecen-1-ol, 1, by a three step procedure in an overall yield of31%. Alkylation of phthalimide with alkanol, 1, under Mitsunobuconditions, followed by reaction with hydrazine gave1-aminoundec-10-ene, 2, in 66% yield. The amine was derivatized withO-acetylsalicyloyl chloride and the resulting alkene, 3, was oxidized tothe acid using potassium permanganate. Removal of the acetate, followedby acid precipitation provided compound XXXI in 47% yield based on amine2. ##STR65##

Delivery Systems

The compositions of the present invention may include one or more activeagents.

In one embodiment, compounds I-CXXIII or poly amino acids or peptidesthat include at least one of these compounds may be used directly as adelivery carrier by simply mixing one or more compound, poly amino acidor peptide with the active agent prior to administration.

In an alternative embodiment, the compounds, poly amino acids, orpeptide may be used to form microspheres containing the active agent.These compounds, poly amino acids, or peptides are particularly usefulfor the oral administration of certain biologically-active agents, e.g.,small peptide hormones, which, by themselves, do not pass or only afraction of the administered dose passes through the gastrointestinalmucosa and/or are susceptible to chemical cleavage by acids and enzymesin the gastrointestinal tract.

If the modified amino acids, poly amino acids, or peptides are to beconverted into microspheres, the mixture is optionally heated to atemperature ranging between about 20 and about 50° C., preferably about40° C., until the modified amino acid(s) dissolve. The final solutioncontains between from about 1 mg and to about 2000 mg of compound, polyamino acid, or peptide per mL of solution, preferably between about 1and about 500 mg per mL. The concentration of active agent in the finalsolution varies and is dependent on the required dosage for treatment.When necessary, the exact concentration can be determined by, forexample, reverse phase HPLC analysis.

When the compounds, poly amino acids, or peptides are used to preparemicrospheres, another useful procedure is as follows: Compounds, polyamino acids, or peptides are dissolved in deionized water at aconcentration ranging between about 75 and about 200 mg/ml, preferablyabout 100 mg/ml at a temperature between about 25° C. and about 60° C.,preferably about 40° C. Particulate matter remaining in the solution maybe removed by conventional means such as filtration.

Thereafter, the compound, poly amino acid, or peptide solution,maintained at a temperature of about 40° C., is mixed 1:1 (V/V) with anaqueous acid solution (also at about 40° C.) having an acidconcentration ranging between about 0.05N and about 2N, preferably about1.7N. The resulting mixture is further incubated at 40° C. for a periodof time effective for microsphere formation, as observed by lightmicroscopy. In practicing this invention, the preferred order ofaddition is to add the compound, poly amino acid, or peptide solution tothe aqueous acid solution.

Suitable acids for microsphere formation include any acid which does not

(a) adversely effect the modified amino acids, poly amino acids, orpeptides e.g., initiate or propagate chemical decomposition;

(b) interfere with microsphere formation;

(c) interfere with microsphere incorporation of the active agent cargo;and

(d) adversely interact with the active agent cargo.

Preferred acids for use in this aspect include acetic acid, citric acid,hydrochloric acid, phosphoric acid, malic acid and maleic acid.

A microsphere stabilizing additive may be incorporated into the aqueousacid solution or into the compound or cargo solution prior to the-microsphere formation process. With some active agents the presence ofsuch additives promotes the stability and/or dispersibility of themicrospheres in solution.

The stabilizing additives may be employed at a concentration rangingbetween about 0.1 and 5% (w/v), preferably about 0.5% (w/v). Suitable,but non-limiting, examples of microsphere stabilizing additives includegum acacia, gelatin, methyl cellulose, polyethylene glycol,polypropylene glycol, carboxylic acids and salts thereof, andpolylysine. The preferred stabilizing additives are gum acacia, gelatinand methyl cellulose.

Under the above conditions, the compound molecules, poly amino acids, orpeptides form hollow or solid matrix type microspheres wherein the cargois distributed in a carrier matrix or capsule type microspheresencapsulating liquid or solid cargo. If the compound, poly amino acid,or peptide microspheres are formed in the presence of a solublematerial, e.g., a pharmaceutical agent in the aforementioned aqueousacid solution, this material will be encapsulated within themicrospheres. In this way, one can encapsulate pharmacologically activematerials-such as peptides, proteins, and polysaccharides as well ascharged organic molecules, e.g., antimicrobial agents, which normallyhave poor bioavailability by the oral route. The amount ofpharmaceutical agent which may be incorporated by the microsphere isdependent on a number of factors which include the concentration ofagent in the solution, as well as the affinity of the cargo for thecarrier. The compound, poly amino acid, or peptide microspheres do notalter the physiological and biological properties of the active agent.

Furthermore, the encapsulation process does not alter thepharmacological properties of the active agent. Any pharmacologicalagent can be incorporated within the microspheres. The system isparticularly advantageous for delivering chemical or biological agentswhich otherwise would be destroyed or rendered less effective byconditions encountered within the body of the animal to which -t isadministered, before the microsphere reaches its target zone (i.e., thearea in which the contents of the microsphere are to be released) andfor delivering pharmacological agents which are poorly absorbed in thegastro-intestinal tract. The target zones can vary depending upon thedrug employed.

The particle size of the microsphere plays an important role indetermining release of the active agent in the targeted area of thegastro-intestinal tract. The preferred microspheres have diametersbetween about≦0.1 microns and about 10 microns, preferably between about0.5 microns and about 5 microns. The microspheres are sufficiently smallto release effectively the active agent at the targeted area within thegastro-intestinal tract such as, for example, between the stomach andthe jejunum. Small microspheres can also be administered parenterally bybeing suspended in an appropriate carrier fluid (e.g., isotonic saline)and injected directly into the circulatory system, intramuscularly orsubcutaneously. The mode of administration selected will vary, ofcourse, depending upon the requirement of the active agent beingadministered. Large amino acid microspheres (>50 microns) tend to beless effective as oral delivery systems.

The size of the microspheres formed by contacting compounds, poly aminoacids, or peptides with water or an aqueous solution containing activeagents can be controlled by manipulating a variety of physical orchemical parameters, such as the pH, osmolarity or ionic strength of theencapsulating solution, size of the ions in solution and by the choiceof acid used in the encapsulating process.

The administration mixtures are prepared by mixing an aqueous solutionof the carrier with an aqueous solution of the active ingredient, justprior to administration. Alternatively, the carrier and the biologicallyor chemically active ingredient can be admixed during the manufacturingprocess. The solutions may optionally contain additives such asphosphate buffer salts, citric acid, acetic acid, gelatin, and gumacacia.

Stabilizing additives may be incorporated into the carrier solution.With some drugs, the presence of such additives promotes the stabilityand dispersibility of the agent in solution.

The stabilizing additives may be employed at a concentration rangingbetween about 0.1 and 5% (W/V), preferably about 0.5% (W/V). Suitable,but non-limiting, examples of stabilizing additives include gum acacia,gelatin, methyl cellulose, polyethylene glycol, carboxylic acids andsalts thereof, and polylysine. The preferred stabilizing additives aregum acacia, gelatin and methyl cellulose.

The amount of active agent is an amount effective to accomplish thepurpose of the particular active agent. The amount in the compositiontypically is a pharmacologically or biologically effective amount.However, the amount can be less than a pharmacologically or biologicallyeffective amount when the composition is used in a dosage unit form,such as a capsule, a tablet or a liquid, because the dosage unit formmay contain a multiplicity of carrier/biologically or chemically activeagent compositions or may contain a divided pharmacologically orbiologically effective amount. The total effective amounts can then beadministered in cumulative units containing, in total, pharmacologicallyor biologically or chemically active amounts of biologically orpharmacologically active agent.

The total amount of active agent, and particularly biologically orchemically active agent, to be used can be determined by those skilledin the art. However, it has surprisingly been found that with somebiologically or chemically active agents, the use of the presentlydisclosed carriers provides extremely efficient delivery, particularlyin oral, intranasal, sublingual, intraduodenal, or subcutaneous systems.Therefore, lower amounts of biologically or chemically active agent thanthose used in prior dosage unit forms or delivery systems can beadministered to the subject, while still achieving the same blood levelsand therapeutic effects.

The amount of carrier in the present composition is a delivery effectiveamount and can be determined for any particular carrier or biologicallyor chemically active agent by methods known to those skilled in the art.

Dosage unit forms can also include any of excipients; diluents;disintegrants; lubricants; plasticizers; colorants; and dosing vehicles,including, but not limited to water, 1,2-propane diol, ethanol, oliveoil, or any combination thereof.

Administration of the present compositions or dosage unit formspreferably is oral or by intraduodenal injection.

The delivery compositions of the present invention may also include oneor more enzyme inhibitors. Such enzyme inhibitors include, but are notlimited to, compounds such as actinonin or epiactinonin and derivativesthereof. These compounds have the formulas below: ##STR66## Derivativesof these compounds are disclosed in U.S. Pat. No. 5,206,384. Actinoninderivatives have the formula: ##STR67## wherein R⁵ is sulfoxymethyl orcarboxyl or a substituted carboxy group selected from carboxamide,hydroxyaminocarbonyl and alkoxycarbonyl groups; and R⁶ is hydroxyl,alkoxy, hydroxyamino or sulfoxyamino group. Other enzyme inhibitorsinclude, but are not limited to, aprotinin (Trasylol) and Bowman-Birkinhibitor.

The compounds and compositions of the subject invention are useful foradministering biologically or chemically active agents to any animalssuch as birds; mammals, such as primates and particularly humans; andinsects. The system is particularly advantageous for deliveringchemically or biologically or chemically active agents which wouldotherwise be destroyed or rendered less effective by conditionsencountered before the active agent its target zone (i.e. the area inwhich the active agent of the delivery composition are to be released)and within the body of the animal to which they are administered.Particularly, the compounds and compositions of the present inventionare useful in orally administering active agents, especially those whichare not ordinarily orally deliverable.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples illustrate the invention without limitation. Allparts are given by weight unless otherwise indicated.

EXAMPLE 1

Compound XIX was prepared as follows:

A 3 L three-neck round bottom flask was fitted with an overheadmechanical stirrer and a thermometer, and the flask was cooled in anice- bath. A solution of 8-aminocaprylic acid (100.0 g, 0.65 moles) in2M aqueous sodium hydroxide (1.4 L) was charged into the round bottomflask. The temperature of the solution was kept at about 5° C., andO-acetylsalicyloyl chloride (198.6 g, 0.76 moles, 1.2 equiv.) was addedportionwise over 7 hours. The mixture was stirred at 5° C. for 12 hoursto yield a yellow homogenous solution. The solution was acidified with1M hydrochloric acid to pH 6.8 and was extracted with ethyl acetate(2×600 mL). The pH of the aqueous layer was readjusted to 6.3 and wasfurther extracted with ethyl acetate (2×600 mL). The organic layers werecombined, dried over anhydrous sodium sulfate, filtered, and evaporatedunder reduced pressure. The residue was redissolved in a minimum volumeof 2M aqueous sodium hydroxide, and the pH of the solution was between9.5 and 10. The mixture was acidified with stirring with 1M hydrochloricacid to pH of about 6.2, and a solid was formed. The solid was filtered,washed with water (3×300 mL), and recrystallized from 55% methanol/water(v/v) to yield Compound XVIII as an off-white solid (99.7 g, 57%).

Properties are listed below.

mp 116°-117° C. ¹ H NMR (300 MHz, DMSO-d₆)δ: 12.70 (1H, br s), 11.95(1H, br s) 8.81 (1H, t), 7.82 (1H, m), 7.381 (1H, m), 6.84 (2H, m), 2.36(2H, q), 2.18 (2H, t), 1.50 (4H, br m), 1.28 (6H, m), Anal. Calcd forC₁₅ H₂₁ NO₄ : C, 64.50; H, 7.58;1N, 5.02.

Found: C, 64.26; H, 7.81;N, 4.93.

Similar procedures were used to prepare Compounds I, II, III, IV, VI,IX, X, XI, XII, XI, XIV, XX, XXI, XXIII, XXVII, XXVIII, XXXIII, andXXXIV.

Properties are listed below.

Compound I: ¹ H NMR (300 MHz, D₂ O): δ1:5 (2H, m) 2.0 (2H, t) 2.3 (2H,t)7.5 (2H, t) 7.6 (1H, m) 7.3 (2H, m)

Compound II: ¹ H NMR (300 MHz, D₂ O): δ1.4 (8H, m) 1.7 (6H, m) 2.1(2H,t) 1.25 (1H, m) 3.05 (2H, t)

Compound III: ¹ H NMR(300 MHz, DMSO-d₆): δ0.7 (3H, m) 0.9 (2H, m) 1.1(3H, q) 1.6 (5H, m) 1.75 (2H, q) 2.1 (2H, t) 3.0 (2H, q) 7.9 (1H, m).

Compound IV: Anal. Calcd for C₁₁ H₁₃ NO₄ : C, 59.9, H, 5.87, N, 6.27Found: C, 58.89, H, 5.85,N, 6.07. ¹ H NMR (300 MHz, DMSO-d₆): δ1.8 (2H,m) 2.3 (2H, t) 3.1 (2H,q) 6.9 (2H, t) 7.4 (1H, t) 7.8 (1H, d) 8.85 (1H,t) 12.0 (1H, s) 12.15 (1H, s)

Compound VI: ¹ H NMR (300 MHz, D₂ O): δ0.8 (2H, m) 1.1 (4H, m) 1.4(2H,q) 1.6 (7H, m) 2.15 (4H, m) 3.1 (2H, t)

Compound IX: ¹ H NMR (300 MHz, DMSO-d₆): δ0.9 (q, 3H), 1.2 (m, 7H), 1.3(q, 2H), 1.5 (q, 3H), 1.9 (d, 2H), 2.0 (d, 1H), 2.2 (t, 2H), 3.0 (q,3H), 7.7 (s, 1H)

Compound X: ¹ H NMR (300 MHz, DMSO-d₆): δ0.7(d, 2H), 0.9 (dd, 1H),1.2-1.3 (m, 7H), 1.5 (q, 3H), 1.6-1.8 (m, 5H), 2.15 (t, 2H), 3.0 (m,3H), 7.5 (s, 1H), 12.0 (s, 1H)

Compound XI: Anal. Calcd for C₁₅ H₂₀ NO₃ Cl: C, 60.48, H, 6.78,N, 4.70Found: C, 60.4, H, 6.68,N, 4.53. ¹ H NMR (300 MHz, DMSO-d₆): 6 1.28 (m,6H) 1.48 (m, 4H) 2.19 (t, 2H) 3.19 (qt, 2H), 7.323-7.48 (m, 4H), 8.39(t, 1H), 12.09 (s, 1H)

Compound XII: Anal. Calcd for C₁₇ H₂₂ NO₃ : C, 66.42, H, 7.23,N, 4.56Found: C, 65.80, H, 7.17,N, 4.14. ¹ H NMR (300 MHz, DMSO-d₆): δ1.25 (m,6H) 1.43-1.49 (m, 4H) 2.18 (t, 2H) 3.15 (qt, 2H), 6.72 (d, 1H),7.21-7.26 (m, 2H), 7.39 (t, 1H), 7.48 (d, 1H), 7.65 (t, 1H), 8.21 (t,1H)

Compound XIII: Anal. Calcd for C₁₅ H₁₉ NO₃ : C, 60.18, H, 6,41,N, 4.67Found: C, 60.26, H, 6.53,N, 4.61. ¹ H NMR (300 MHz, DMSO-d₆): δ1.28 (m,6H), 1.45-1.52 (m, 4H), 2.19 (t, 2H), 2.22 (qt, 2H), 7.13 (m, 2H),7.43-7.53 (m, 1H), 8.67 (t, 1H)12.03 (s, 1H)

Compound XIV: Anal. Calcd for C₁₄ H₂₀ N₂ O₃ :·0.66 H₂ O: C, 63.04, H,7.91,N. 10.34 Found: C, 63.21, 7.59, 10.53 ¹ H NMR (300 MHz, DMSO-d₆):δ1.22-12.8 (m, 6H), 1.48-1.50 (m, 4H), 2.18 (t, 2H), 3.24 (qt, 2H), 7.48(m, 1H), 8.15 (d, 1H), 8.63-8.69 (m, 2H), 8.97 (d, 1H)

Compound XX: Anal. Calcd for C₁₅ H₂₀ NO₃ F: C, 60.09, H, 7.19,N, 4.98Found: C, 63.82, H, 7.23,N, 4.94. ¹ H NMR (300 MHz, DMSO-d₆): δ1.28 (m,6H) 1.49 (m, 4H) 2.19 (t, 2H) 3.23 (qt, 2H), 7.24-7.30 (m, 2H),7.49-7.60 (m, 2H), 11.99 (s, 1H)

Compound XXI: Anal. Calcd for C₁₇ H₂₃ NO₄ : C, 66.85, H, 7.61,N, 4.58Found: C, 66.81, H, 7.69,N, 4.37. ¹ H NMR (300 MHz, DMSO-d₆): δ1.26 (m,6H) 1.42-1.50 (m, 4) 2.18 (t, 2H) 3.13 (qt, 2H), 6.63 (d, 1H), 6.80 (t,1H), 6.86 (d, 1H), 7.15 (t, 1H), 7.39 (d, 1H), 7.60 (d, 1H), 8.03 (t,1H), 9.95 (s, 1H), 12.12 (s, 1H)

Compound XXIII: Anal. Calcd for C₁₅ H₂₇ NO₃ : C, 66.86, H, 10.22,N, 5.19Found: C, 66.92, H, 10.72, N, 5.14. ¹ H NMR (300 MHz, DMSO-d₆):δ1.56-1.34 (m, 13H) 1.46 (t, 2H) 1.60-1.68 (m, 5H), 2.04 (t, 1H), 2.17(t, 2H), 2.97 (qt, 2H), 7.62 (t, 1H), 11.98 (s, 1H)

Compound XXVII: Anal. Calcd for C₁₈ H₂₇ NO₄ : C, 67.25, H, 8.48,N, 4.36Found: C, 67.23, H, 8.57,N, 4.20. ¹ H NMR (300 MHz, DMSO-d₆): δ1.22-1.26(m, 12H) 1.45-1.51 (m, 4H) 2.16 (t, 2H) 3.25 (qt, 2H), 6.85 (t, 2H),7.37 (t, 1H), 7.81 (d, 1H), 8.79 (t, H), 11.95 (s, 1H), 12.72 (s,1 H)

Compound XXVIII: ¹ H NMR (300 MHz, DMSO-d₆): δ1.26 (8H, br m), 1.49 (4H,m), 2.17 (2H, t), 3.26 (2H, m), 6.86 (2H, m), 7.37 (1H, m), 7.83 (1H,m), 8.80 (1H, t), 11.95 (1H, s), 12.73 (1H, s).

Compound XXXIII: ¹ H NMR (300 MHz, DMSO-d₆): δ1.2 (a, 2H), 1.3 (q,2H),1.3 (q, 2H), 1.5 (q, 2H), 2.2 (t, 2H), 3.0 (q, 2H), 3.5 (s, 2H), 7.3 (m,5H), 8.0 (s, 1H)

Compound XXXIV: Anal. Calcd for C₁₂ H₁₇ NO₄ : C, 62.23, H, 6.83,N, 5.57Found: C, 61.93, H, 6.80,N, 5.56. ¹ H NMR (300 MHz, DMSO-d₈): δ1.24-1.34(m, 2H) 1.49 1.57 (m, 4H) 2.19 (t, 2H) 3.26 (qt, 2H), 6.68 (t, 2H), 7.37(s, 1H), 7.83 (d, 1H) 8.81 (t, 1H), 12.08 (s, 1H), 12.72 (s, 1 H)

EXAMPLE 1A

An alternate synthesis of compound XIX was as follows:

A 5 L three-neck round bottom flask was fitted with a heating mantle, anoverhead mechanical stirrer, an addition funnel, and a thermometer. Thereaction was performed under an argon atmosphere.Hydroxylamine-O-sulfonic acid (196.7 g, 1.74 moles, 1.10 equiv.) andformic acid (1 L) were charged into the round bottom flask and stirredto form a white slurry. A solution of cyclooctanone (200.0 g 1.58 moles,1.0 equiv.) in formic acid (600 mL) was added dropwise to the whiteslurry via the addition funnel. After the addition, the addition funnelwas replaced by a reflux condenser, and the reaction was heated toreflux (internal temperature about 105° C.) for 1 hour to give a brownsolution. After the solution was cooled to room temperature, it waspoured into a mixture of saturated aqueous ammonium chloride (1.5 L) andwater (1.5 L). The aqueous mixture was extracted with chloroform (3×1200mL). The combined chloroform layers were transferred into a beaker, andsaturated sodium bicarbonate (2 L) was added slowly. The chloroformlayer was then separated, dried over anhydrous sodium sulfate, andevaporated under reduced pressure to afford a brown oil. The oil wasplaced in a 500 mL round bottom flask with a magnetic stirrer. The roundbottom flask was placed in a silicon oil bath and was fitted with ashort path vacuum distillation head equipped with a thermometer. ACow-type receiver was connected to three 250 mL flasks.2-Azacyclononanone (145 g, 65%, mp 64°-69° C.) was obtained by vacuumdistillation (fraction with head temperature range from 80°- to 120° Cat pressures between 3.0 and 3.4 mmHg).

A 5 L three-neck round bottom flask was fitted with a heating mantle, anoverhead mechanical stirrer, a reflux condenser, and a 29 thermometer. Asuspension of 2-azacyclononanone (83 g, 0.59 moles, 1.0 equiv.) in 5Maqueous sodium hydroxide (650 mL, 3.23 moles, 5.5 equiv.) was chargedinto the round bottom flask. The mixture was heated to reflux (internaltemperature about 110° C.) for 4 hours to yield a clear yellow,solution. The heating mantle and reflux condenser were removed. Afterthe solution cooled to room temperature, it was diluted with water (650mL) and cooled further in an ice bath. Finely ground O-acetylsalicyloylchloride (1 14.7 g, 0.59 moles, 1.0 equiv.) was added portionwise to thesolution with stirring and continued cooling over 1 hour. After anadditional 30 minutes, the ice-bath was removed and stirring wascontinued at ambient temperature for 21 hours to give a brownish yellowsolution. The stirred mixture was acidified with 2M sulfuric acid (about850 mL) to a pH of about 1, and a yellow solid was formed. The solid wascollected by filtration and was dissolved in warm methanol (1.7 L).Activated charcoal (about 5 g) was added to the methanol, and thesolution was stirred for 10 minutes. The activated charcoal was removedby filtration, and the charcoal residue was washed with additional 300mL methanol. Water (2 L) was added to the combined filtrates (i.e. the 2L methanol), and an off-white solid precipitated upon standing at 4° C.overnight. The crude product was filtered and was recrystallized from65% methanol/water (v/v) to yield Compound XIX (69.1 g, 42%) asoff-white solid.

Properties are listed below:

mp 116°-117° C.; HPLC, ¹ H NMR and Anal. Calcd for C₁₅ H₂₁ NO₄ : C,64.50; H, 7.58;N, 5.02. Found: C, 64.26; H, 7.81;N, 4.93.

EXAMPLE 2

Compound XXXI was prepared as follows:

1-Aminoundec-10-ene. A mixture of 10-undecene-1-ol (5.00 g, 29.36 mmol,1 equiv), triphenylphosphine (7.70 g, 29.36 mmol, 1 equiv) andphthalimide (4.32 g, 29.36 mmol, 1 equiv) in dry tetrahydrofuran (THF,30 mL) was stirred vigorously under argon. Diethyl azodicarboxylate(DEAD, 5.11 g, 29.36 mmol, 1 equiv) was diluted with THF (12 mL) andadded dropwise by syringe. After the addition, the reaction was stirredat room temperature for 4 hours. The solvent was evaporated under vacuumand ether (30 mL) was added to precipitate the triphenylphosphine oxideand hydrazine dicarboxylate which were removed by filtration. Theprecipitate was rinsed with ether (2×30 mL) and the combined filtrateswere evaporated to afford a yellow solid. The yellow solid wastriturated with warm hexanes (3×50 mL) and filtered. The combinedhexanes were evaporated to give 1-phthalimidylundec-10-ene as a yellowwax.

The yellow wax was dissolved in an ethanolic solution (38 mL) ofhydrazine hydrate (1.47 g, 1 equiv, 29.36 mmol). The mixture was heatedat reflux for 2 hours. After the mixture was cooled to room temperature,concentrated hydrochloric acid (30 mL) was added and the solid wasfiltered through a sintered glass filter. The residue was washed withwater (50 mL) and the combined filtrates were evaporated to provide ayellow solid. The yellow solid was redissolved in 1M NaOH (100 mL) andextracted with ether (2×50 mL). The ether was dried and evaporated toprovide a yellow oil.

The oil was purified by Kugelrohr distillation (ca. 0.1 mmHg, 100° C.)to provide 1-aminoundec-10-ene (2) as a light yellow oil (3.29 g, 66%).

Properties are listed below.

¹ H NMR (300 mHz, DMSO-d₆); δ1.23 (14H, br m), 1.99 (2H, m), 2.48 (2H,m), 4.94 (2H, m), 5.77 (1H, m).

1-(O-Acetylsalicyloylamino)undec-10-ene. O-Acetylsalicyloyl chloride(3.82 g, 19.25 mmol, 1 equiv) in THF (30 mL) was cooled in an ice bath.Triethylamine (1.95 g, 19.25 mmol, 1 equiv), followed by1-aminoundec-10-ene (3.26 g, 19.25 mmol, 1 equiv) in THF (10 mL) wereadded via syringe. The ice bath was removed and the reaction was stirredat room temperature for 3.5 hours. After removal of the solvent, theresidue was dissolved in EtOAc (50 mL) and washed with water (2×30 mL).The organic layer was dried and evaporated to afford1-(O-acetylsalicyloyl-amino)undec-10-ene as a colorless oil, in aquantitative yield, 6.59 g.

Properties are listed below.

¹ H NMR (300 mHz, DMSO-d₆ : δ1.26 (12H, br s), 1.47 (2H,m), 1.99(2H,m),2.19 (3H,s), 3.15 (2H, q), 4.95 (2H, m), 5.78 (1H, m), 7.15 (1H, m),7.30 (1H, m), 7.50 (2H, m) 8.24 (1H, t).

Compound XXXI

1-(O-Acetylsalicyloylamino)under-10-ene (6.59 g, 19.25 mmol, 1 equiv) indichloromethane (108 mL) was added to a mixture of water (108 mL),sulfuric acid (9M, 13 mL), glacial acetic acid (2.16 mL) andmethyltrialkyl(C₈ -C₁₀)ammonium chloride (0.32 g) (Adogen® 464,available from Aldrich Chemical Co.). The mixture was stirred vigorouslyin an ice bath and potassium permanganate (9.13 g, 57.75 mmol, 3 equiv)was added in portions over 1.5 hours. After the addition, the ice bathwas removed and the resultant purple solution was stirred at roomtemperature for 20 hours. The solution was cooled in an ice bath andsodium bisulfite (6.8 g) was added to dissipate the excess permanganate.The organic layer was separated and the aqueous layer was extracted withethyl acetate (2×50 mL). The combined organic layers were washed withbrine (50 mL), dried and evaporated. Sodium hydroxide (2M, 50 mL) wasadded to the residue and stirred for 30 min. The solution was dilutedwith water (50 mL), washed with ether (50 mL) and acidified to pH 1 with2M hydrochloric acid. A solid formed and was collected by filtration.Recrystallization of the solid from 65% MeOH/H₂ O gave XXXI as a tansolid (2.78 g, 47% based on the amine).

Properties are listed below.

¹ H NMR (300 mHz, DMSO-d₆): δ1.24 (10H, br m), 1.51 (4H, m), 2.17 (2H,t), 3.27 (2H, m), 6.86 (2H, m), 7.37 (1H m), 7.82 (1H, m), 8.80 (1H, t),11.95 (1H, s), 12.72 (1H, s).

EXAMPLE 3

Compound LXXXVI was prepared as follows:

A one liter three-neck round bottom flask was fitted with a magneticstirrer and a condenser. A solution of 3-(4-aminophenyl)propionic acid(30 g, 0.182 moles) in methylene chloride (300 mL) was charged to theflask and trimethylsilyl chloride (46.2 mL, 0.364 moles) was added inone portion. The reaction mixture was refluxed for 1.5 hours, allowed tocool to room temperature, and then immersed in an ice/water bath.Triethylamine. (76.2 mL, 0.546 moles) ws added, followed by2-methoxycinnamoyl chloride (35.8 g, 0.182 moles). The reaction mixturewas allowed to warm to room temperature and then stirred for 48 hours.The solvent was removed by rotary evaporation and saturated sodiumbicarbonate solution and ethyl acetate were added to the residue. Thelayers were separated, the aqueous layer was acidified to pH 1.4 with 2Naqueous sulfuric acid and extracted with ethyl acetate (2×400 mL). Thecombined organic extracts were concentrated in vacuo and the residuerecrystallized from 50% (v/v) aqueous methanol to provide the product asa tan solid (48.57 g, 82%).

Properties are listed below.

¹ H NMR (300 MHz, DMSO-d₆): δ12.1 (1H, br), 7,8 (1H, dd), 7.6 (3H, m),7.4 (1H, m), 7.3 (2H, m), 7.1 (1H, d), 7.0 (1H, t), 6.9 (1H, d), 3.9(3H, s), 2.8 (2H, t), 2.5 (4H, m).

Anal. Calcd for C₁₉ H₁₉ NO₄ : C, 70.14; H, 5.88;N, 4.31. Found: C,69.76; H, 5.91;N, 4.21.

EXAMPLE 4

Compound CXVII was prepared as follows:

A 3 L three-neck round bottom flask was fitted with an overheadmechanical stirrer and a thermometer. A solution of 8-aminocaprylic acid(10.0 g, 0.054 moles) in 2M aqueous sodium hydroxide (1.4 L) was chargedinto the round bottom flask and O-nitrobenzoyl chloride (12.0 g, 0.065moles, 1.2 equiv.) was added portionwise over 7 h. The mixture wasstirred at 25° C. for 12 h to afford a yellow homogenous solution. Thesolution was acidified with 1M hydrochloric acid to about pH 2, an oilyresidue separated and was decanted. The oil was dissolved in stirredwater (300 mL) and cooled in and ice/water bath. The productprecipitated as a white solid. The solid was filtered, washed with water(3×300 mL), and recrystallized from 55% acetonitrile/water (v/v) toprovide Compound CXVII as an off-white solid (7.4 g, 47%). mp 89°-92° C.

Properties are listed below.

¹ H NMR (300 MHz, DMSO-d₆) δ: 12.0 (1H, s), 8.65 (1H, t), 8.0 (1H, dd),7.8 (1H, rn), 7.65 (1H, m), 7.5 (1H, m), 3.2 (2H, q), 2.2 (2H, t), 1.5(4H, br m), 1.3 (6H, br m).

Anal. Calcd for C₁₅ H₂₀ N₂ O₅ : C, 58.41; H, 6.54;N, 9.09. Found: C,58.50; H, 6.71;N, 9.14.

The other compounds of the invention can be readily prepared byfollowing the procedures described in Examples 1-4.

EXAMPLES 5-15 In Vivo Evaluation of Recombinant Growth Hormone in Rats

Dosing compositions were prepared by mixing the modified amino acids andrecombinant human growth hormone (rhGH) as listed in Table 1 below in aphosphate buffer solution at a pH of about 7-8.

Rats were administered the dosing composition by sublingual, oralgavage, intraduodenal administration, or colonic administration.Delivery was evaluated by using an ELISA assay for rhGH from MedixBiotech, Inc. For intracolonic administration, a sample was prepared anddosed to fasted rats at 25 mg/kg of carrier in a buffered solutioncontaining propylene glycol (0-50%) and 1 mg/kg rhGH.

Results are illustrated in Table 1 below.

Comparative Example 5A

rhGH (6 mg/ml) was administered by oral gavage to a rat, and deliverywas evaluated according to the procedure of Example 5.

Results are illustrated in Table 1 below.

                                      TABLE 1    __________________________________________________________________________    In Vivo Delivery of rhGH                                  Mean Peak Serum               Carrier Dose                      Drug Dose                           Method of                                  Levels of rhGH    Example          Carrier               (mg/kg)                      (mg/kg)                           Administration                                  (ng/mL)    __________________________________________________________________________    5     I    500    6    oral     26.6 +/- 43.83    5A    none 0      6    oral    <.10 +/- 10    6     V    500    6    oral    3.22 +/- 7.2    7     VI   500    6    oral    19.34 +/- 18.73    8     VIII 500    6    oral    73.41 +/- 70.3    9     IX   500    6    oral    28.70 +/- 41.7    10    XIII 25     1    colonic                                  109.52 +/- 36.1    11    XIX  200    3    oral    60.92 +/- 26.3    12    XIX  25     1    colonic                                  111.52 +/- 16.4    13    XIX  100    3    sublingual                                  119.14 +/- 65.6    14    XIX  25     1    intranasal                                   92.7 +/- 73.2    15    XXVII               25     1    colonic                                  73.72 +/- 4.9    __________________________________________________________________________

EXAMPLES 16-27 In Vivo Evaluation of Recombinant Growth Hormone in Rats

Preparation of Dosing solutions.

The delivery agents were reconstituted with distilled water and adjustedto pH 7.2-8.0 with either aqueous hydrochloric acid or aqueous sodiumhydroxide. A stock solution of rhGH was prepared by mixing rhGH,D-mannitol and glycine and dissolving this mixture in 2% glycerol/water.The stock solution was then added to the delivery agent solution.Several delivery agent to active agent ratios were studied.

In vivo experiments.

Male Sprague-Dawley rats weighing 200-250 g were fasted for 24 hours andadministered ketamine (44 mg/kg) and chlorpromazine (1.5 mg/kg) 1 5minutes prior to dosing. The rats were administered one of the dosingsolutions described above by subcutaneous injection, intranasalinstillation, or sublingual instillation. Blood samples were collectedserially from the tail artery for serum calcium concentrationdetermination or serum rhGH concentrations. The dose of rhGHadministered in these experiments was 0.1 mg/kg.

Serum rhGH concentrations were quantified by an rhGH enzyme immunoassaytest kit. The results are given in Table 2 and FIGS. 1 and 2.

In FIG. 2 the circles represent the response following SL dosing of anaqueous solution of compound CXXIII and rhGH. The squares represent theresponse following IN dosing of an aqueous solution of compound CXXIIIand rhGH. The triangles represent the response following IC dosing of anaqueous solution of compound CXXIII and rhGH. The dose of compoundCXXIII was 25 mg/kg and the dose of rhGH was 1 mg/kg.

Comparative Example 16A

rhGH (1 mg/kg) was administered by oral gavage to a rat, and deliverywas evaluated according to the procecure of Example 16.

Results are illustrated in Table 2 below.

                  TABLE 2    ______________________________________    Delivery Agent Enhancement of Recombinant Human Growth Hormone    (rhGH) Bioavailability Administered by Subcutaneous Administration.                      Delivery Agent Dose                                    Peak Serum  rhGH!    Example           Deliver Ageny                      (mg/kg)       (ng/mL)    ______________________________________    16     CXXIII     1.0           22 ± 3    16A    None       0.0            4 ± 2    17     CXXIII     2.5           25 ± 5    18     CXXIII     25            30 ± 6    19     CXI        2.5           16 ± 2    20     LVIII      1.0            29 ± 10    21     LXXXVI     1.0           22 ± 7    22     LXXXVI     2.5           23 ± 5    23     LXI        2.5           26 ± 5    24     CX         1.0           15 ± 3    25     CXV        1.0           25 ± 3    26     LXVI       1.0           33 ± 5    27     CIX        1.0           16 ± 3    ______________________________________

EXAMPLES 28-33 In Vivo Evaluation of Interferon in Rats

Dosing compositions were prepared by mixing the modified amino acidcompounds and interferon a2b as listed in Table 3 below in a Trizma®hydrochloride buffer solution (Tris-HCl) at a pH of about 7-8. Propyleneglycol (0-25%) was added as a solubilizing agent, if necessary.

Rats were administered the dosing composition by oral gavage,intraduodenal administration, or intracolonic administration. Deliverywas evaluated by use of an ELISA assay for human interferon a fromBiosource, Inc.

Results of intracolonic administration are illustrated in Table 3 below.

Comparative Example 28A

Interferon a2b (250 μg/kg) was administered intracolonically to rats,and delivery was evaluated according to the procedure of Example 14.

Results are illustrated in Table 3 below.

                  TABLE 3    ______________________________________    In Vivo Delivery of Interferon by Intracolonic Administration                                      Mean Peak Serum                   Carrier Dose                             Interferon Dose                                      Levels of Interferon    Example           Carrier (mg/kg)   (μg/kg)                                      (pg/mL)    ______________________________________    28     VII     100       250      5241 +/- 2205    28A    none    0         250      0    29     XI      100       250      1189 +/- 1373    30     XII     100       250      6955 +/- 2163    31     XIX     100       250      11193 +/- 8559    32     XXI     100       250      4238 +/- 2789    33     XXXIV   100       250      4853 +/- 5231    ______________________________________

Results are illustrated in Table 4 below.

EXAMPLES 34-37 In Vivo Evaluation of Salmon Calcitonin in Rats

Dosing compositions were prepared by mixing the modified amino acids andsalmon calcitonin as listed in Table 4 below. 400 mg of carrier wereadded to 2.9 mL of 25% aqueous propylene glycol. The resultant solutionwas stirred, and the pH was adjusted to 7.2 with sodium hydroxide(1.0N). Water was added to bring the total volume to 2.0 mL. The samplehad a final carrier concentration of 200 mg/mL. Calcitonin (10 μg) wasadded to the solution. The total calcitonin concentration was 2.5 μg/ml.

For each sample a group of fasted rats were anesthetized. The rats wereadministered the dosing composition by oral gavage, intracolonicinstillation, or intraduodenal administration. Blood samples werecollected serially from the tail artery. Serum calcium was determined bytesting with a Calcium Kit (Sigma Chemical Company, St. Louis, Mo.,USA).

Results are illustrated in Table 4 below.

                                      TABLE 4    __________________________________________________________________________    In Vivo Delivery of Calcitonin                                  Maximum Decrease              Carrier Dose                     Drug Dose                          Method of                                  in Serum Calcium.    Example          Carrier              (mg/kg)                     (μg/kg)                          Administration                                  (% below baseline)    __________________________________________________________________________    34    I   400    10   oral    35    V   400    10   oral    18.35 +/- 2.87    36    XIX 10     3    intracolonic                                  26.49 +/- 12.3    37    XIX 200    7.5  oral    25.48 +/- 4.7    __________________________________________________________________________

EXAMPLES 38-43 In Vivo Evaluation of Salmon Calcitonin in RatsPreparation of Dosing solution.

The delivery agents were reconstituted with distilled water and adjustedto pH 7.2-8.0 with either aqueous hydrochloric acid or aqueous sodiumhydroxide. A stock solution of sCT was prepared by dissolving sCT incitric acid (0.085N). The stock solution was then added to the deliveryagent solution. Several different delivery agent to active agent ratioswere studied.

In vivo experiments.

Male Sprague-Dawley rats weighing 200-250 g were fasted for 24 hours andadministered ketamine (44 mg/kg) and chlorpromazine (1.5 mg/kg) 1 5minutes prior to dosing. The rats were administered one of the dosingsolutions described above by subcutaneous injection. Blood samples werecollected serially from the tail artery for serum calcium concentration.

Serum calcium concentrations were quantified by the o-cresolphthaleincomplexone method (Sigma) using a UV/VIS spectrophotometer (PerkinElmer). The results are given in Table 5.

EXAMPLE 38A

Salmon calcitonin was administered by oral gavage to rats, and deliverywas evaluated according to the Procedure of Example 38. The results aregiven in Table 5 below.

                  TABLE 5    ______________________________________    Delivery Agent Enhancement of Salmon Calcitonin (sCT, dosed    at 0.2 μg/kg) Bioavailability Administered by    Subcutaneous Administration.                      Delivery Agent Dose                                    Percent Decrease in    Example           Deliver Ageny                      (μg/kg)    Serum Calcium    ______________________________________    38     CXXIII     2             17 ± 3    38A    None       0             17 ± 2    39     CXXIII     20            25 ± 4    40     CXXIII     200           25 ± 5    41     CXXIII     2000          26 ± 5    42     CXI        20            21 ± 4    43     CXIV       20            20 ± 3    ______________________________________

EXAMPLES 44-50 In Vivo Evaluation of Heparin in Rats

Dosing compositions were prepared by mixing the modified amino acids andheparin as listed in Table 4. In a test tube, 900 mg of carrier weredissolved in 3 mL of propylene glycol, and 0.299 g of sodium heparin wasdissolved in 3 mL of water. The solutions were mixed by vortex. Sodiumhydroxide (10M) was added to the resulting mixture until a solution wasobtained. The pH was then adjusted to 7.4+/-0.5 with concentratedhydrochloric acid, and the final solution was sonicated at 40° C. for 30minutes.

A group of fasted, conscious rats were administered the dosingcompositions by oral gavage. Blood samples were collected by cardiacpuncture following the administration of ketamine (44 mg/kg)., Heparinactivity was determined by utilizing the activated partialthromboplastim time (APTT) according to the method of Henry, J. B.,Clinical Diagnosis and Management by Laboratory Methods; Philadelphia,Pa.; W B Saunders (1979).

Results are illustrated in Table 6 below.

Comparative Example 44A

Heparin (100 mg/kg) was administered by oral gavage to rats, and heparinactivity was determined according to the procedure of Example 44.

                  TABLE 6    ______________________________________    In Vivo Delivery of Heparin by Oral Administration                    Carrier Dose                               Drug Dose                                      Mean Peak APTT    Example Carrier (mg/kg)    (mg/kg)                                      (sec)    ______________________________________    44      II      300        100    25.45 +/- 2.8    44A     none    none       100     20.7 +/- 0.17    45      III     300        100    38.64 +/- 17    46      V       300        100     87.4 +/- 34.1    47      XII     300        100     49.53 +/- 17.1    48      XIX     300        100    119.99 +/- 56.3    49      XXXI     50        25      127.56 +/- 22.97    50      XXXI     50        10     50.85 +/- 9.1    ______________________________________

EXAMPLE 51

The method of Example 44 was followed, substituting low molecular weightheparin for the heparin and varying the amounts of propylene glycol andwater for solubilization as, necessary.

EXAMPLES 50-58 In vivo Evaluation of Parathyroid Hormone in Rats

Preparation of dosing solutions.

The delivery agents were reconstituted with distilled water and/orpropylene glycol and adjusted to an apparent pH of 7.2-8.0 with eitheraqueous hydrochloric acid or aqueous sodium hydroxide. A stock solutionof parathyroid hormone was prepared by dissolving parathyroid hormone inwater. The parathyroid hormone solution was then added to the deliveryagent solution. Several different delivery agent to active agent ratioswere studied.

In vivo experiments.

Male Sprague-Dawley rats weighing 200-250 g were fasted for 24 hours andadministered ketamine (44 mg/kg) and chlorpromazine (1.5 mg/kg) 15minutes prior to dosing. The rats were administered one of the dosingsolutions described above by oral gavage or intracolonic instillation.Blood samples were collected serially from the tail artery for serumdetermination of parathyroid hormone concentration. Serum parathyroidhormone concentrations were quantified by a parathyroid hormoneradioimmunoassay test kit.

In vivo Oral administration.

Oral administration of solutions containing parathyroid hormone (PTH)and the non-a-amino acid delivery agents was tested in vivo in rats.

The result show a significant increase in the orai bioavailability ofparathyroid hormone as compared to similar administration of the activeagent alone.

Data are presented in Table 7.

                  TABLE 7    ______________________________________    Delivery Agent Enhancement of Parathyroid Hormone (PTH) Oral    Bioavailability.                                            Peak                   Carrier          Active  Serum    Exam-          Dose    Method of                                    Agent Dose                                             PTH!    ple   Carrier  mg/kg   Administration                                    (μg/kg)                                            (pg/mL)    ______________________________________    51    CXXIII   100     intracolonic                                    25      130 ± 20    52    CXXIII   250     oral     100     75 ± 25    53    CXXIII   250     oral     25      20 ± 6    54    CVIII    100     intracolonic                                    25      115 ± 20    55    LXXXVI   100     intracolonic                                    25      40 ± 12    56    LVIII    100     intracolonic                                    25      145 ± 25    57    CXIV     100     intracolonic                                    25      65 ± 15    58    LXXXIX   100     intracolonic                                    25      70 ± 15    ______________________________________

The above mentioned patents, applications, test methods, andpublications are hereby incorporated by reference in their entirety.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed description. Allsuch obvious variations are within the full intended scope of theappended claims.

What is claimed is:
 1. A composition comprising:(A) at least one activeagent; and (B) a compound having the following formula ##STR68## or asalt thereof.
 2. A composition as defined in claim 1, wherein saidactive agent is selected from the group consisting of a biologicallyactive agent, a chemically active agent, and a combination thereof.
 3. Acomposition as defined in claim 2, wherein said biologically activeagent comprises at least one peptide, mucopolysaccharide, carbohydrate,or lipid.
 4. A composition as defined in claim 2, wherein saidbiologically active agent comprises heparin.
 5. A composition as definedin claim 2, wherein said biologically active agent comprises lowmolecular weight heparin.
 6. A composition comprising:(a) at least oneactive agent; and (b) a carrier comprising a compound having thefollowing formula: ##STR69## or a salt thereof, wherein said carrier isselected from the group consisting of a poly(amino acid) and apolypeptide.
 7. A composition as defined in claim 6, wherein saidcarrier comprises a polypeptide.
 8. A dosage unit form comprising:(A) acomposition as defined in claim 1; and (B) (a) an excipient (b) adiluent, (c) a disintegrant, (d) a lubricant, (e) a plasticizer, (f) acolorant, (g) a dosing vehicle, or (h) any combination thereof.
 9. Adosage unit form as defined in claim 8, wherein said active agent isselected from the group consisting of a biologically active agent, achemically active agent, and a combination thereof.
 10. A dosage unitform as defined in claim 9, wherein said active agent is selected fromthe group consisting of at least one peptide, mucopolysaccharide,carbohydrate, and a combination thereof.
 11. A dosage unit form asdefined in claim 9 wherein said biologically active agent comprisesheparin.
 12. A dosage unit form as defined in claim 9 wherein saidbiologically active agent comprises low molecular weight heparin.
 13. Adosage unit form as defined in claim 8, comprising a tablet, a capsule,or a liquid.
 14. A dosage unit form as defined in claim 13, wherein saiddosing vehicle is selected from the group consisting of water, 1,2-diol,ethanol, and any combination thereof.
 15. A method for administering abiologically-active agent to an animal in need of said agent, saidmethod comprising administering orally to said animal a composition asdefined in claim
 2. 16. A compound having the formula ##STR70## or asalt thereof.
 17. A method for preparing a composition, said methodcomprising mixing:(A) at least one active agent; (B) at least onecompound as defined in claim 16; and (C) optionally, a dosing vehicle.18. The composition as defined in claim 3, wherein said biologicallyactive agent is selected from the group consisting of human growthhormone, bovine growth hormone, growth hormone-releasing hormone, aninterferon, interleukin-II, insulin, heparin, low molecular weightheparin, calcitonin, erythropoietin, atrial naturetic factor, anantigen, a monoclonal antibody, somatostatin, adrenocorticotropin,gonadotropin releasing hormone, oxytocin, vasopressin, cromolyn sodium,vancomycin, desferrioxamine (DFO), and any combination thereof.
 19. Acomposition as defined in claim 18, wherein said biologically activeagent comprises an interferon, interleukin-II, insulin, heparin, lowmolecular weight heparin, calcitonin, oxytocin, vasopressin, vancomycin,DFO, or combinations thereof.
 20. A composition as defined in claim 2,wherein said biologically active agent comprises recombinant humangrowth hormone.
 21. The dosage unit form as defined in claim 10, whereinsaid biologically active agent is selected from the group consisting ofhuman growth hormone, bovine growth hormone, growth hormone-releasinghormone, an interferon, interleukin-II, insulin, heparin, low molecularweight heparin, calcitonin, erythropoietin, atrial naturetic factor, anantigen, a monoclonal antibody, somatostatin, adrenocorticotropin,gonadotropin releasing hormone, oxytocin, vasopressin, cromolyn sodium,vancomycin, desferrioxamine (DFO), and any combination thereof.
 22. Adosage unit form as defined in claim 21, wherein said biologicallyactive agent comprises an interferon, interleukin-II, insulin, heparin,low molecular weight heparin, calcitonin, oxytocin, vasopressin,vancomycin, DFO, or combinations thereof.
 23. A dosage unit form asdefined in claim 9, wherein said biologically active agent comprisesrecombinant human growth hormone.